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R E S E A R C H Open Access

Vitamin A decreases pre-receptor amplificationof glucocorticoids in obesity: study on the effectof vitamin A on 11beta-hydroxysteroiddehydrogenase type 1 activity in liver andvisceral fat of WNIN/Ob obese ratsVara Prasad SS Sakamuri1, Prashanth Ananthathmakula1, Giridharan Nappan Veettil2 and

Vajreswari Ayyalasomayajula1*

Abstract

Background: 11b-hydroxysteroid dehydrogenase type 1 (11b-HSD1) catalyzes the conversion of inactive

glucocorticoids to active glucocorticoids and its inhibition ameliorates obesity and metabolic syndrome. So far, no

studies have reported the effect of dietary vitamin A on 11b-HSD1 activity in visceral fat and liver under normal

and obese conditions. Here, we studied the effect of chronic feeding of vitamin A-enriched diet (129 mg/kg diet)

on 11b-HSD1 activity in liver and visceral fat of WNIN/Ob lean and obese rats.

Methods: Male, 5-month-old, lean and obese rats of WNIN/Ob strain (n = 16 for each phenotype) were divided

into two subgroups consisting of 8 rats of each phenotype. Control groups received stock diet containing 2.6 mg

vitamin A/kg diet, where as experimental groups received diet containing 129 mg vitamin A/Kg diet for 20 weeks.

Food and water were provided ad libitum. At the end of the experiment, tissues were collected and 11b-HSD1

activity was assayed in liver and visceral fat.Results: Vitamin A supplementation significantly decreased body weight, visceral fat mass and 11b-HSD1 activity in

visceral fat of WNIN/Ob obese rats. Hepatic 11b-HSD1 activity and gene expression were significantly reduced by

vitamin A supplementation in both the phenotypes. CCAAT/enhancer binding protein a (C/EBPa), the main

transcription factor essential for the expression of 11b-HSD1, decreased in liver of vitamin A fed-obese rats, but not

in lean rats. Liver receptor a (LXRa), a nuclear transcription factor which is known to downregulate 11b-HSD1

gene expression was significantly increased by vitamin A supplementation in both the phenotypes.

Conclusions: This study suggests that chronic consumption of vitamin A-enriched diet decreases 11b-HSD1

activity in liver and visceral fat of WNIN/Ob obese rats. Decreased 11b-HSD1 activity by vitamin A may result in

decreased levels of active glucocorticoids in adipose tissue and possibly contribute to visceral fat loss in these

obese rats. Studying the role of various nutrients on the regulation of 11b-HSD1 activity and expression will help in

the evolving of dietary approaches to treat obesity and insulin resistance.

* Correspondence: [email protected] of Biochemistry, National Institute of Nutrition, Indian Council

of Medical Research, Jamai Osmania PO, Hyderabad-500 604, Andhra

Pradesh, India

Full list of author information is available at the end of the article

Sakamuri et al. Nutrition Journal 2011, 10:70

http://www.nutritionj.com/content/10/1/70

2011 Sakamuri et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the CreativeCommons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, andreproduction in any medium, provided the original work is properly cited.
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Background11b-hydroxysteroid dehydrogenase type 1 (11b-HSD1)

modulates the local glucocorticoid metabolism by cata-

lyzing the conversion of inactive glucocorticoids (corti-

sone in humans and 11-dehydrocorticosterone in

rodents) to active glucocorticoids (cortisol in humans

and corticosterone in rodents). Recent studies have

shown that, 11b-HSD1 plays an important role in the

development of obesity [1,2]. Transgenic mice overex-

pressing 11b-HSD1 in adipose tissue developed visceral

obesity [3], where as 11b-HSD1 knock-out mice dis-

played resistance to diet-induced obesity [4]. Adminis-

tration of carbenoxolone, a non-selective 11b-HSD1

inhibitor, has been shown to decrease obesity in animal

models [5]. 11b-HSD1 gene expression is directly regu-

lated by CCAAT/enhancer-binding protein a (C/EBPa)

[6], where as its expression is down regulated by Liver

receptor a (LXRa) [7].Studies on rodent models of obesity provided valuable

information regarding the molecular mechanisms

involved in the development of obesity and its asso-

ciated complications. WNIN/Ob obese rat colony was

established by selective breeding of spontaneously

mutated obese rat, identified in 80 year-old inbred wis-

tar rat colony at National Institute of Nutrition (NIN,

Hyderabad, India) [8]. The rat colony shows three phe-

notypes (also genotypes), lean (+/+), carrier (+/-) and

obese (-/-). WNIN/Ob lean and carrier rats are fertile,

where as obese rats are sterile. The obese rat colony is

maintained by crossing between carrier rats, whichresults in genotypic ratio (and also phenotypic ratio) of

1:2:1 (lean: carrier: obese) indicating the autosomal co-

dominant nature of the mutation. WNIN/Ob obese rats

develop severe obesity with onset around 35 days of age.

These rats are hyperphagic, hyperinsulinemic, hyperlep-

tinemic and dyslipidemic [8]. Preliminary studies on

WNIN/Ob obese rats have revealed no molecular

defects in leptin and leptin receptor. Recent studies have

located the mutation on 5th chromosome [unpublished

data] and further investigations are underway to identify

the molecular mutation leading to the development of

obesity in these obese rats.

Diet plays an important role in the development, pro-gression and amelioration of chronic diseases like obe-

sity and diabetes. Studies on the effect of nutrients on

11b-HSD1 activity and expression will greatly help in

understanding the mechanisms underlying the develop-

ment of obesity and its associated metabolic disorders.

Vitamin A, an important micronutrient has divergent

biological functions in animal physiology. Vitamin A-

enriched diet has been shown to decrease adiposity and

alter adipose tissue gene expression in rats [9]. Our pre-

vious studies on supplementation of high but non-toxic

dose of vitamin A to obese rats of WNIN/Ob strain

have shown to decrease adiposity [10,11]. So far, no stu-

dies have addressed the role of vitamin A in the regula-

t io n o f 1 1b-HSD1 activity in normal and obese

conditions. As vitamin A is known to alter the expres-

sion of genes regulated by C/EBPa [12], here, we

hypothesize that supra-physiological (high but non-

toxic) dose of vitamin A modulates 11b-HSD1 activity

in adipose tissue under normal and obese conditions.

To test our hypothesis, we studied the activity of 11b-

HSD1 in visceral fat and liver of WNIN/Ob lean and

obese rats, after chronic challenging with vitamin A-

enriched diet (129 mg/Kg diet) for 20 weeks.

MethodsAnimal experiment

Male, 5-month-old WNIN/Ob lean and obese rats (n =

16 for each phenotype) were obtained from the NationalCentre for Laboratory animal sciences, and divided into

two groups A and B based on phenotype. Each group

was further divided into two subgroups (AI, AII & BI,

BII) consisting of 8 rats from each phenotype. The ani-

mals were housed individually in a temperature (22 2

C) and light controlled (12 h cycle) animal facility. Ani-

mals of AI and BI subgroups were fed on stock diet

containing 2.6 mg of vitamin A/Kg diet (Recommended

daily allowance), while those in AII and BII subgroups

were fed on experimental diet containing 129 mg of

vitamin A/Kg diet (as retinyl palmitate- a generous gift

from Nicholas Piramal India Limited). Animals were

maintained on their respective diets for a period of 20

weeks. Food and water were provided ad libitum. Daily

food intake and weekly body weights were recorded.

The study was approved by Institutional Animal Ethical

Committee (IAEC). At the end of the experiment, blood

was collected after overnight fast, between 9.00 AM and

11.00 AM. Animals were sacrificed and tissues were col-

lected and stored at -80C for further analysis.

Plasma and tissue parameters

Plasma corticosterone levels were measured by radioim-

munoassay (RIA) kit method (Siemens, Los Angeles,

USA). Plasma, liver and visceral fat retinol levels weremeasured by methods described previously [10].

Measurement of 11b-HSD1 activity

11b-HSD1 functions as a reductase in vivo, reactivating

corticosterone from inactive 11-dehydrocorticosterone.

However, in tissue homogenates, dehydrogenase activity

predominates, therefore, 11b-HSD1 activity was measured

by the conversion of corticosterone to 11-dehydrocorticos-

terone. We studied 11b-HSD1 activity in visceral fat by

taking omental adipose tissue, as it is known to exhibit



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higher enzyme activity. Post-nuclear fractions from liver

and visceral fat were prepared by centrifuging tissue

homogenate at 1000 g for 20 min. 11b-HSD1 activity was

measured in post nuclear fractions of liver and visceral fat

by incubating in duplicate at 37C in Krebs-Ringer buffer

containing glucose (0.2%), NADP (1 mM) and 1, 2, 6, 7-

[3H4] corticosterone (50 nM). Conditions were optimized

for both tissues to ensure first order kinetics, by adjusting

protein concentrations for liver (40 g/ml) and omental

adipose tissue (1 mg/ml). After incubation (40 min for

liver and 6 h for omental adipose tissue), steroids were

extracted with ethyl acetate, the organic phase was evapo-

rated under nitrogen, and extracts resuspended in mobile

phase (50% water, 30% acetonitrile and 20% methanol).

Steroids were separated by HPLC using reverse phase C18

column and fractions containing corticosterone and 11-

dehydrocorticosterone were collected and radioactivity

was counted in liquid scintillation counter (PerkinElmer,USA).

Measurement of glycerol-3-phosphate dehydrogenase

(GPDH) activity

50 mg of liver tissue was homogenized in 10 mM Tris-

Hcl buffer (pH-7.4) containing 1 mM 2-mercaptoetha-

nol and 5% protease inhibitor cocktail (Sigma, USA).

Homogenate was centrifuged at 10,000 g for 20 minutes

at 4C and the supernatant was used for the estimation

of cytosolic GPDH activity. GPDH activity was mea-

s ured b y the metho d o f White and Kaplan and

expressed as nanomoles of NADH utilized/min/mg of

protein (13).

Measurement of relative gene expression by semi-

quantitative Reverse Transcription PCR

As lean rats have limited omental adipose tissue, which

we utilized for the enzyme assay, we carried out gene

expression work only in liver but not in adipose tissue.

Total cellular RNA from liver was extracted using the

method of Chomczynski and Sacchi [14]. The integrity

of RNA was checked using 1% agarose gels stained with

ethidium bromide. Total RNA was quantified by spec-

trophotometric absorption at 260 nm. 1.0 g of RNA

was used for synthesizing first strand cDNA. Thereverse transcription (RT) reaction was carried out by

incubating RNA with 0.5 g oligo dT primer (Sigma,

USA) and 100 units of Molony murine leukemia virus

reverse transcriptase (Finnzymes, Espoo, Finland) at 37

C for 60 min. Total reaction volume used for RT was

20 L. An aliquot of cDNA was amplified in a 20 L

reaction mixture. PCR conditions are given below: dena-

turation at 94C for 1 minute, annealing at 60-64C for

45 sec and polymerization for 70 C for 1 min with

DyNAzyme II DNA polymerase (Finnzymes, Espoo, Fin-

land). A final extension was carried out at 70 C for 7

min. The amount of RNA and the annealing tempera-

ture for different genes were standardized for linearity.

Sequences of primers used for amplification are 11b-

HSD1: forward primer-5-GAGGAAGGGCTCCAG-3and reverse primer-5-GAGCAAACTTGCTTGCA-

3(NM_017080), LXRa: forward primer-5-GCCCCATG-

GACACCTA-3 and reverse primer-5-TGAGGGTCG

GGTGCAA-3 (NM_031627), C/EBPa: forward primer-

5-GAGCCGAGATAAAGCCAA-3 and reverse primer

5-CTTTCAGGCGACACCA-3 (NM_012524), b-actin:

forward primer-5- ACCAACTGGGACGACATGGA-3and reverse primer-5- TCTCAAACATGATCTGGG

TCA-3 (NM_031144). b-actin mRNA was amplified as

an internal control. Number of amplification cycles for

each gene was standardized so that amplification will be

in the logarithmic phase. After amplification, 8 L of

reaction mixture were electrophoresed on agarose gel

(2%) in Tris-acetate EDTA buffer (pH 8.2). The ethi-dium bromide stained bands were visualized by UV-

transilluminator and analyzed densitometrically using

Quantity One software (Bio-Rad, version 4.4.0).

Immunoblotting

Nuclear protein was extracted from liver as described

previously [15,16]. Protein content was estimated by

Bradford method. Proteins were separated by 10% SDS-

PAGE gel and probed by specific antibodies raised

against C/EBPa and LXRa (Santa Cruz Biotechnology,

Inc., and Santa Cruz, CA). Equal loading of protein and

transfer were ensured by staining membranes with Pon-

seau S. Bands were scanned using GS 710 densitometer

(Biorad, CA, USA) and band densities were analyzed by

Quantity one software (Biorad, version 4.4.0).

Statistics

Data were analyzed by one way ANOVA by using least

significant difference (LSD) post hoc test. Data are pre-

sented as mean S.E. Statistical significance was taken

at P < 0.05 level (two tailed).

ResultsEffect of vitamin A supplementation on physical and

biochemical parametersVitamin A supplementation significantly reduced body

weight, body weight gain and visceral fat mass in obese

rats compared to stock diet-fed obese rats (Table 1).

Vitamin A supplementation did not alter the visceral fat

mass or body weight in lean rats as compared with their

stock-diet fed counterparts (Table 1). Food intake was

not affected by vitamin A supplementation in both the

phenotypes as compared with their respective control

groups (Table 1). Consumption of vitamin A-enriched

diet for 20 weeks significantly increased retinol levels in

liver and visceral fat of lean and obese rats as compared



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with their respective stock-diet fed rats (Table 2).

Plasma corticosterone levels were not altered by vitamin

A supplementation in both the phenotypes (Table 2).

Effect of vitamin A supplementation on 11b-HSD1 activity

in visceral fat

Vitamin A supplementation significantly decreased 11b-

HSD1 activity in visceral fat of obese rats as compared

to stock diet-fed obese rats (Figure 1). In contrast to the

observation in obese rats, vitamin A supplementation

significantly increased 11b-HSD1 activity in visceral fat

of lean rats as compared with their stock diet-fed lean

counterparts (Figure 1).

Effect of vitamin A supplementation on hepatic 11b-HSD1

activity and expression

Vitamin A supplementation significantly decreased 11b-

HSD1 activity in the liver of lean and obese rats as

against their stock diet-fed lean and obese counterparts

(Figure 2). In line with the decreased enzyme activity,

11b-HSD1 mRNA levels were significantly lower in high

vitamin A-fed lean and obese rats as compared with their

stock diet-fed lean and obese counterparts (Figure 2).

Effect of vitamin A supplementation on C/EBPa, LXRa

mRNA and protein levels in liver

To explain the possible mechanism involved in the vita-

min-A mediated downregulation of 11b-HSD1 in liver,

we studied the hepatic expression of C/EBPa and LXRa

both at mRNA and protein levels. Vitamin A supple-

mentation significantly increased the hepatic C/EBPa

gene expression in both the phenotypes (Figure 3) as

compared with their respective control rats. However,

elevated hepatic C/EBPa protein levels were observed

only in vitamin A- supplemented lean rats, where as in

obese rats, hepatic C/EBPa protein levels were signifi-

cantly low (Figure 3). Hepatic LXRa mRNA and protein

levels significantly decreased by feeding of vitamin A

enriched diet to WNIN/Ob lean and obese rats as com-

pared with their respective stock diet fed-lean and obese

counterparts (Figure 4).

Effect of vitamin A supplementation on hepatic glycerol-

3-phospahte dehydrogenase (GPDH) activity

As vitamin A supplementation decreased hepatic 11b-

HSD1 activity in lean and obese rats, we studied the

activity of cytosolic glycerol-3-phosphate dehydrogenase(GPDH), which is induced by glucocorticoids in hepato-

cytes (17). In line with the decreased 11b-HSD1 activity,

GPDH activity was also significantly decreased by vita-

min A supplementation in both the phenotypes as com-

pared with their respective controls (Figure 5).

DiscussionIn this communication, we report the impact of chronic

feeding of vitamin A-enriched diet on 11b-HSD1 activity

in liver and visceral fat of WNIN/Ob obese rats, a new

genetic rat model of obesity. Here, we demonstrate that

Table 1 Effect of vitamin A supplementation on food

intake and physical parameters in WNIN/Ob lean and

obese rats

AI AII BI BII

Pre-treatment body

weight (g)

375 16 362 25 628 38 590 11

Post-treatment bodyweight (g)

406 23 452 15 916 48 778 19*

Body weight (g) 91 20 89 14 285 19 188 14#

Dai ly food intake (g) 18 0.6 18 0.8 26 1.4 26 0.9

Visceral fat (g/100 gbody.wt)

2.3 0.4 2 .5 0.4 10.5 0.7 7 .0 0.5#

Values are mean S.E of 8 rats. Values with # and * mark are significant at P

< 0.01 and < 0.05 level respectively (by one-way ANOVA). Comparisons were

made between normal and high vitamin A-fed groups of each phenotype

(AI-Lean: 2.6 mg vitamin A/Kg diet, AII-Lean: 129 mg vitamin A/Kg diet,

BI-Obese: 2.6 mg vitamin A/kg diet and BII-Obese: 129 mg vitamin A/kg diet)

Table 2 Effect of vitamin A supplementation on

biochemical parameters in WNIN/Ob lean and obese rats

AI AII BI BII

Plasma corticosterone(ng/mL)

227 50 170 15 256 27 287 34

Plasma retinol (g/dL) 28 0.5 28 0.5 40 0.6 43 2.0

Liver total retinol(mg/g tissue)

0.9 0.1 9.8 0.4## 0.4 0.02 8.1 0.3##

Visceral fat retinol(g/g tissue)

4.0 1.2 41 2.7## 2.0 0.1 4.8 2.0##

Values are means S.E of 8 rats. Mean values with## mark are significant at P

< 0.001 level (by one-way ANOVA). Comparisons were made between normal

and high vitamin A-fed groups of each phenotype (AI-Lean: 2.6 mg vitamin

A/kg diet, AII-Lean: 129 mg vitamin A/kg diet, BI-Obese: 2.6 mg vitamin A/kg

diet and BII-Obese: 129 mg vitamin A/kg diet).

Figure 1 Effect of vitamin A supplementation on adipose

tissue 11b-HSD1 activity in WNIN/Ob lean and obese rats .Values are means S.E for 8 rats. Mean values with * mark are

significant at P 0.05 level (by one-way ANOVA). Comparisons were

made between normal and high vitamin A-fed groups of each

phenotype (AI-Lean: 2.6 mg vitamin A/kg diet, AII-Lean: 129 mg

vitamin A/kg diet, BI-Obese: 2.6 mg vitamin A/kg diet and BII-Obese:

129 mg/kg diet).



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feeding of vitamin A-enriched diet to WNIN/Ob obese

rats decreases body weight, fat mass and 11b-HSD1

activity in liver and visceral fat. To our knowledge, this

is the first study to link vitamin A and pre-receptor

metabolism of glucocorticoids in normal and obese

conditions.

Retinoic acid, one of the metabolically-active forms of

vitamin A, regulates cel lular gene express ion through

retinoic acid receptors (RARs) and retinoid receptors

(RXRs). Adipose tissue is a good target organ for vita-

min A action, as it stores significant amount of vitamin

A and expresses RAR and RXR transcription factors.

Retinoic acid is known to inhibit preadipocyte differen-

tiation [18] and remodel white adipose tissue to brown

adipose tissue [19]. Retinaldehyde, another functional

form of vitamin A is reported to decrease adipogenesis

and ameliorate diet-induced obesity in rodent models

[20]. In line with our previous observation, high (but

not toxic) dose of vitamin A decreased visceral fat mass

and bodyweight in obese rats [10]. Previous studies have

reported that cortisol (corticosterone in rodents) is

essential for preadipocyte differentiation [21] and adi-

pose-specific overexpression of 11b-HSD1 in mice,

results in increased preadiocyte differentiation [3]. As

reported previously, WNIN/Ob obese rats have exhib-

ited higher 11b-HSD1 activity in visceral fat than their

lean counter parts [22]. In the present study, vitamin A

significantly decreased 11b-HSD1 activity in visceral fat,

which is associated with significant decrease in visceral

fat mass. Vitamin A mediated visceral fat loss in obese

rats could be due to decreased preadipocyte differentia-

tion, as reduced 11b-HSD1 activity results in decreased

tissue corticosterone levels. In contrast to the observa-

tion in obese rats, vitamin A-challenged lean rats had

increased 11b-HSD1 activity in visceral fat, however,

increased enzyme activity had no impact on visceral fat

mass. The differential effects of vitamin A on 11b-HSD1

activity and visceral fat mass in lean and obese rats

could be due to other physiological factors that are

altered in obese condition.

Figure 2 (A) Effect of vitamin A supplementation on hepatic 11b-HSD1 activity in WNIN/Ob lean and obese rats . Values are means S.Efor 8 rats. Mean values with * mark are significant at P 0.05 level (by one-way ANOVA). (B) Effect of vitamin A supplementation on 11b-HSD1

gene expression in liver of WNIN/Ob lean and obese rats. Quantified densitometric values are expressed relative of 1 for lean control rats (AI). (C)

Representative photo of 11b-HSD1 PCR product, stained by ethidium bromoide. (D) Representative photo of PCR b-Actin product (internal

control), stained by ethidium bromide. Comparisons were made between normal and high vitamin A-fed groups of each phenotype (AI-Lean:

2.6 mg vitamin A/kg diet, AII-Lean: 129 mg vitamin A/kg diet, BI-Obese: 2.6 mg vitamin A/kg diet and BII-Obese: 129 mg/kg diet).



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Vitamin A may regulate the 11b-HSD1 activity, by

altering its gene expression through direct and indirect

mechanisms. Presently, retinoic acid-response elements

are not reported on 11b-HSD1 promoter to support the

direct effect of vitamin A on 11b-HSD1 gene expression.C/EBPa has been shown to be obligatory for the expres-

sion of 11b-HSD1 [4]. Previous studies have reported

that retinoic acid downregulates the expression of genes

like resistin that are regulated by C/EBPa in adipose tis-

sue [13]. Vitamin A-mediated regulation of 11b-HSD1

gene expression in adipose tissue may be mediated by

indirect mechanism through C/EBPa.

11b-HSD1 plays an important role in the regulation of

carbohydrate and lipid metabolism in liver. 11b-HSD1-

KO mice have improved insulin sensitivity [4], where as

transgenic overexpression of 11b-HSD1 in liver results

in the development of impaired glucose tolerance [23].

WNIN/Ob obese rats have lower hepatic 11b-HSD1

expression and activity as observed in other obese

rodent models [22]. In the present study, vitamin A sup-

plementation at high doses decreased hepatic 11b-HSD1activity and expression in both lean and obese pheno-

types. Supporting to our hypothesis, activity of GPDH, a

glucocorticoid inducible enzyme, was also decreased in

vitamin A supplemented lean and obese rats. Based on

these observations, it is possible that vitamin A may reg-

ulate the expression of hepatic glucocorticoid target

genes by regulating the expression of 11b-HSD1.

To understand the possible mechanisms involved in

the downregulation of 11b-HSD1 in liver, we studied

the expression of C/EBPa at mRNA and protein levels.

In our study, vitamin A supplementation increased

Figure 3 (A) Effect of vitamin A supplementation on hepatic C/EBPa mRNA and protein levels in WNIN/Ob lean and obese rats.Quantified densitometric values are expressed relative of 1 for lean control rats (AI). (B) Representative photo of C/EBPa PCR product, stained by

ethidium bromoide. (C) Representative photo ofb-Actin PCR product (internal control), stained by ethidium bromide. (D) Quantified

densitometric values from the western blot are expressed relative of 1 for A-I (lean) as control. (E) Representative Western blot of hepatic C/EBPa

protein. (F) Ponseau stained western blot. Values are means SE for 3-4 rats at P 0.05 level. Comparisons were made between normal and

high vitamin A-fed groups of each phenotype (AI-Lean: 2.6 mg vitamin A/kg diet, AII-Lean: 129 mg vitamin A/kg diet, BI-Obese: 2.6 mg vitamin

A/kg diet and BII-Obese: 129 mg/kg diet).



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hepatic C/EBPa gene expression in vitamin A-treated

lean and obese rats. In contrast to increased mRNA

levels, C/EBPa protein levels decreased in vitamin A-

supplemented obese rats, suggesting the role of post-

transcriptional regulatory mechanisms. Possibly, vitaminA-mediated downregulation of hepatic 11b-HSD1 is

mediated through different mechanisms in lean and

obese rats, in other words, vitamin A-mediated down

regulation of hepatic 11b-HSD1 may be C/EBPa-depen-

dent in obese rats, while it is independent of C/EBPa in

lean rats.

Previous studies have reported that LXRa ligands

down regulate 11b-HSD1 through indirect mechanisms

[7]. In this study, vitamin A supplementation increased

hepatic LXRa mRNA and protein levels in WNIN/Ob

lean and obese rats. In support to the elevated LXRa

Figure 4 (A) Effect of vitamin A supplementation on hepatic LXRa mRNA and protein levels in WNIN/Ob lean and obese rats.Quantified densitometric values are expressed relative of 1 for AI (lean) as control. (B) Representative photo of LXRa PCR product, stained by

ethidium bromoide. (C) Representative photo ofb-Actin PCR product (internal control), stained by ethidium bromide. (D) Quantified

densitometric values from the western blot are expressed relative of 1 for lean control rats (AI). (E) Representative Western blot of hepatic LXR a

protein. (F) Ponseau stained western blot. Values are means SE for 3-4 rats at P 0.05 level. Comparisons were made between normal and

high vitamin A-fed groups of each phenotype (A I-Lean: 2.6 mg vitamin A/kg diet, A II-Lean: 129 mg vitamin A/kg diet, B I-Obese: 2.6 mg

vitamin A/kg diet and B II-Obese: 129 mg/g diet).

Figure 5 Effect of vitamin A supplementation on hepatic GPDH

activity in WNIN/Ob lean and obese rats. Activity was reported as

nmoles of NADH utilized/min/mg protein. Values are means SE

for 8 rats at P 0.05 level. Comparisons were made between normal

and high vitamin A-fed groups of each phenotype (AI-Lean: 2.6 mg

vitamin A/kg diet, AII-Lean: 129 mg vitamin A/kg diet, BI-Obese: 2.6

mg vitamin A/kg diet and BII-Obese: 129 mg/kg diet).



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gene expression, ABCA1 (ATP-binding cassette trans-

porter protein 1), a classical target gene of LXRa is ele-

vated in lean and obese rats (unpublished data). Vitamin

A-mediated downregulation of hepatic 11b-HSD1 gene

expression is may be mediated through LXRa. Another

mechanism, through which dietary vitamin A may down

regulate 11b-HSD1, is by providing high concentration

of RXR ligand, 9-cis-retinoic acid. LXRa forms heterodi-

mer with RXR and this step is essential for LXRa-

mediated gene transcription. Higher concentration of

RXR ligand may result in the increased recruitment of

LXRa:RXR heterodimers to 11b-HSD1 gene promoter

resulting in decreased 11b-HSD1 gene expression.

11b-HSD1 gene expression in liver and adipose tissue

is also regulated by various hormones and cytokines

present in the plasma [24]. It is possible that Vitamin

A-mediated alterations in the levels of these signaling

molecules may also affect the 11b-HSD1 activity in liverand adipose tissue of WNIN/Ob lean and obese rats.

ConclusionsIn summary, we showed for the first time that supra-

physiological dose of vitamin A through diet decreases

11b-HSD1 activity in visceral fat and liver of WNIN/Ob

obese rat. The observed vitamin A-mediated reduction

in 11b-HSD1 activity in the visceral fat of obese rats

may contribute to the decreased visceral fat mass in this

model. Further research is needed to understand the

mechanisms involved in the regulation of 11b-HSD1 by

various nutrients in tissues like liver and visceral fat in

order to develop appropriate dietary interventions to

prevent the development of obesity and insulin

resistance.

List of abbreviations used

11-HSD1: 11-hydroxysteroid dehydrogenase type 1; C/EBP: CCAAT/

Enhancer-binding protein ; LXR: Liver receptor ; RXR: Retinoid

receptor; RAR: Retinoic acid receptor; HPLC: High-performance liquidchromatography; SDS-PAGE: Sodium dodecyl sulphate- polyacrylamide gel

electrophoresis.

Acknowledgements

We are grateful to Dr.B.Sesikeran, Director, of the Institute for constant

encouragement during the conduct of the study. Mr.S.S.S.V.Prasad thanks the

Council for Scientific and Industrial Research (CSIR), India for the award of aresearch fellowship to carryout this work.

Author details1Department of Biochemistry, National Institute of Nutrition, Indian Council

of Medical Research, Jamai Osmania PO, Hyderabad-500 604, Andhra

Pradesh, India. 2National Center for Laboratory Animal Sciences, IndianCouncil of Medical Research, Jamai Osmania PO, Hyderabad-500 604, Andhra

Pradesh, India.

Authors contributionsSSSVP carried out plasma corticosterone and 11-HSD1 assays, gene

expression work, data analysis and prepared the first draft. PA carried out

the animal experiment, immunoblotting work, estimated retinol, GPDH

activity, data analysis and manuscript editing. GNV was responsible for

breeding and supply of animals. VA hypothesized, designed and supervisedthe experiment. VA reviewed, finalized the draft and also gave permission to

submit manuscript. All authors read and approved the content of the

manuscript.

Competing interests

The authors declare that they have no competing interests.

Received: 2 August 2010 Accepted: 23 June 2011

Published: 23 June 2011

References

1. Livingstone DEW, Jones G, Smith K, Jamieson PM, Andrew R Kenyon CJ,

Walker BR: Understanding the role of glucocorticoids in obesity: tissue-

specific alterations of corticosterone metabolism in obese zucker rats.

Endocrinology2000, 141:560-563.

2. Rask E, Olsson T, Soderberg S, Andrew R, Livingstone DE, Johnson O,

Walker BR: Tissue-specific dysregulation of cortisol metabolism in human

obesity. J Clin Endocrinol Metab 2001, 86:1418-1421.

3. Masuzaki H, Paterson J, Shinyama H, Morton NM, Mullins JJ, Seckl JR,Flier JS: A transgenic model of visceral obesity and the metabolic

syndrome. Science 2001, 294:2166-2170.

4. Morton N, Paterson J, Masuzaki H, Holmes MC, staels B, Fieevet C, Walker B,Flier J, Mullins J, Seckl : Reduced 11-Hydroxystweroid dehydrogenase

type 1-mediated intra-adipose glucocorticoid regeneration: a novel

protective adaptation to and treatment for the metabolic syndrome.

Program& Abstracts of the 85th Annual Meeting of the Endocrine Society

2003, 114, June 19-22, abstract OR39-5.

5. Nuotio-Antar Alli M, Hachey David L, Hasty Alyssa H: Carbenoxolone

treatment attenuates symptoms of metabolic syndrome and

atherogenesis in obese, hyperlipidemic mice. Am J Physiol Endocrinol

Metab 2007, 293:E1517-E1528.

6. Williams LJ, Lyons V, MaLeod I, Rajan V, Darlington GJ, Poli V, Seckl JR,

Chapman KE: C/EBP regulates hepatic transcription of 11-

Hydroxystweroid dehydrogenase type 1. A novel mechanism for cross-

talk between the C/EBP and glucocorticoid signaling pathways. J Biol

Chem 2000, 275:30232-30239.7. Stuling TM, Oppermann U, Steffensen KR, Schuster GU, Gustafsson JA: Liver

receptors down regulate 11-Hydroxystweroid dehydrogenase type 1

expression and activity. Diabetes 2002, 51:2426-2433.8. Giridharan NV, Harishankar N, Satyavani M: A new rat model for the study

of obesity. Scand J Lab Anim Sci 1996, 23:131-137.

9. Kumar Monica V, Sunvold Gregory D, Scarpace Philip J: Dietary vitamin A

supplementation in rats: suppression of leptin and induction of UCP1

mRNA. J Lip Res 1999, 40:824-29.

10. Jeyakumar SM, Vajreswari A, Giridharan NV: Chronic dietary vitamin A

supplementation regulates obesity in an obese mutant WNIN/Ob rat

model. Obesity2006, 14:52-59.

11. Jeyakumar SM, Vajreswari A, Giridharan NV: Vitamin A supplementation

induces adipose tissue loss through apoptosis in lean and but not inobese rats of WNIN/Ob strain. J Mol Endocrinol 2005, 35:391-398.

12. Felipie Francisco, Luisa Bonet M, Ribot Joan, Palou Andreu: Modulation of

Resistin Activity by Retinoic Acid and vitamin A status. Diabetes 2004,

53:882-89.

13. White H, Kaplan N: Purification and properties of two types of nucleotide

linked glycerol-3-phosphate dehydrogenase from chicken breast muscle

and chicken liver. J Biol Chem 1969, 244:6031-6039.14. Chomczyski P, Sacchi N: Single step method of RNA isolation by acid

guanidinium thiocyanate phenol chloroform extraction. Anal Biochem

1987, 162:156-9.

15. Qiao L, Maclean PS, Schaack J, Orlicky DJ, Darimont C, Pagliassotti M,

Friedman JE, Shao J: C/EBP regulates human adiponectin gene

transcription through an intronic enhancer. Diabetes 2005,

54:1744-1754.

16. Shao J, Qiao L, Janssen RC, Pagliassotti M, Friedman JE: Chronichyperglycemia enhances PEPCK gene expression and hepatocellular

glucose production via elevated liver activating protein/liver inhibitory

protein ratio. Diabetes 2005, 54:976-984.17. Mayer Robert D, Preston Sheryl L, McMorris F Arthur: Glycerol-3-phosphate

dehydrogenase is induced by glucocorticoids in hepatocytes and

hepatoma cells in vitro. J Cell Physiol 2005, 114(2):203-208.



Page 8 of 9
http://-/?-http://www.ncbi.nlm.nih.gov/pubmed/10650936?dopt=Abstracthttp://www.ncbi.nlm.nih.gov/pubmed/10650936?dopt=Abstracthttp://www.ncbi.nlm.nih.gov/pubmed/11238541?dopt=Abstracthttp://www.ncbi.nlm.nih.gov/pubmed/11238541?dopt=Abstracthttp://www.ncbi.nlm.nih.gov/pubmed/11739957?dopt=Abstracthttp://www.ncbi.nlm.nih.gov/pubmed/11739957?dopt=Abstracthttp://www.ncbi.nlm.nih.gov/pubmed/21698810?dopt=Abstracthttp://www.ncbi.nlm.nih.gov/pubmed/21698810?dopt=Abstracthttp://www.ncbi.nlm.nih.gov/pubmed/21698810?dopt=Abstracthttp://www.ncbi.nlm.nih.gov/pubmed/21698810?dopt=Abstracthttp://www.ncbi.nlm.nih.gov/pubmed/21698810?dopt=Abstracthttp://www.ncbi.nlm.nih.gov/pubmed/21698810?dopt=Abstracthttp://www.ncbi.nlm.nih.gov/pubmed/17878220?dopt=Abstracthttp://www.ncbi.nlm.nih.gov/pubmed/17878220?dopt=Abstracthttp://www.ncbi.nlm.nih.gov/pubmed/17878220?dopt=Abstracthttp://www.ncbi.nlm.nih.gov/pubmed/10906322?dopt=Abstracthttp://www.ncbi.nlm.nih.gov/pubmed/10906322?dopt=Abstracthttp://www.ncbi.nlm.nih.gov/pubmed/10906322?dopt=Abstracthttp://www.ncbi.nlm.nih.gov/pubmed/10906322?dopt=Abstracthttp://www.ncbi.nlm.nih.gov/pubmed/10906322?dopt=Abstracthttp://www.ncbi.nlm.nih.gov/pubmed/12145154?dopt=Abstracthttp://www.ncbi.nlm.nih.gov/pubmed/12145154?dopt=Abstracthttp://www.ncbi.nlm.nih.gov/pubmed/12145154?dopt=Abstracthttp://www.ncbi.nlm.nih.gov/pubmed/12145154?dopt=Abstracthttp://www.ncbi.nlm.nih.gov/pubmed/12145154?dopt=Abstracthttp://www.ncbi.nlm.nih.gov/pubmed/16493122?dopt=Abstracthttp://www.ncbi.nlm.nih.gov/pubmed/16493122?dopt=Abstracthttp://www.ncbi.nlm.nih.gov/pubmed/16493122?dopt=Abstracthttp://www.ncbi.nlm.nih.gov/pubmed/16493122?dopt=Abstracthttp://www.ncbi.nlm.nih.gov/pubmed/16216918?dopt=Abstracthttp://www.ncbi.nlm.nih.gov/pubmed/16216918?dopt=Abstracthttp://www.ncbi.nlm.nih.gov/pubmed/16216918?dopt=Abstracthttp://www.ncbi.nlm.nih.gov/pubmed/16216918?dopt=Abstracthttp://www.ncbi.nlm.nih.gov/pubmed/15047602?dopt=Abstracthttp://www.ncbi.nlm.nih.gov/pubmed/15047602?dopt=Abstracthttp://www.ncbi.nlm.nih.gov/pubmed/15047602?dopt=Abstracthttp://www.ncbi.nlm.nih.gov/pubmed/4310830?dopt=Abstracthttp://www.ncbi.nlm.nih.gov/pubmed/4310830?dopt=Abstracthttp://www.ncbi.nlm.nih.gov/pubmed/4310830?dopt=Abstracthttp://www.ncbi.nlm.nih.gov/pubmed/2440339?dopt=Abstracthttp://www.ncbi.nlm.nih.gov/pubmed/2440339?dopt=Abstracthttp://www.ncbi.nlm.nih.gov/pubmed/15919796?dopt=Abstracthttp://www.ncbi.nlm.nih.gov/pubmed/15919796?dopt=Abstracthttp://www.ncbi.nlm.nih.gov/pubmed/15919796?dopt=Abstracthttp://www.ncbi.nlm.nih.gov/pubmed/15919796?dopt=Abstracthttp://www.ncbi.nlm.nih.gov/pubmed/15793235?dopt=Abstracthttp://www.ncbi.nlm.nih.gov/pubmed/15793235?dopt=Abstracthttp://www.ncbi.nlm.nih.gov/pubmed/15793235?dopt=Abstracthttp://www.ncbi.nlm.nih.gov/pubmed/15793235?dopt=Abstracthttp://www.ncbi.nlm.nih.gov/pubmed/15793235?dopt=Abstracthttp://www.ncbi.nlm.nih.gov/pubmed/15793235?dopt=Abstracthttp://www.ncbi.nlm.nih.gov/pubmed/15793235?dopt=Abstracthttp://www.ncbi.nlm.nih.gov/pubmed/15793235?dopt=Abstracthttp://www.ncbi.nlm.nih.gov/pubmed/15919796?dopt=Abstracthttp://www.ncbi.nlm.nih.gov/pubmed/15919796?dopt=Abstracthttp://www.ncbi.nlm.nih.gov/pubmed/2440339?dopt=Abstracthttp://www.ncbi.nlm.nih.gov/pubmed/2440339?dopt=Abstracthttp://www.ncbi.nlm.nih.gov/pubmed/4310830?dopt=Abstracthttp://www.ncbi.nlm.nih.gov/pubmed/4310830?dopt=Abstracthttp://www.ncbi.nlm.nih.gov/pubmed/4310830?dopt=Abstracthttp://www.ncbi.nlm.nih.gov/pubmed/15047602?dopt=Abstracthttp://www.ncbi.nlm.nih.gov/pubmed/15047602?dopt=Abstracthttp://www.ncbi.nlm.nih.gov/pubmed/16216918?dopt=Abstracthttp://www.ncbi.nlm.nih.gov/pubmed/16216918?dopt=Abstracthttp://www.ncbi.nlm.nih.gov/pubmed/16216918?dopt=Abstracthttp://www.ncbi.nlm.nih.gov/pubmed/16493122?dopt=Abstracthttp://www.ncbi.nlm.nih.gov/pubmed/16493122?dopt=Abstracthttp://www.ncbi.nlm.nih.gov/pubmed/16493122?dopt=Abstracthttp://www.ncbi.nlm.nih.gov/pubmed/12145154?dopt=Abstracthttp://www.ncbi.nlm.nih.gov/pubmed/12145154?dopt=Abstracthttp://www.ncbi.nlm.nih.gov/pubmed/12145154?dopt=Abstracthttp://www.ncbi.nlm.nih.gov/pubmed/10906322?dopt=Abstracthttp://www.ncbi.nlm.nih.gov/pubmed/10906322?dopt=Abstracthttp://www.ncbi.nlm.nih.gov/pubmed/10906322?dopt=Abstracthttp://www.ncbi.nlm.nih.gov/pubmed/17878220?dopt=Abstracthttp://www.ncbi.nlm.nih.gov/pubmed/17878220?dopt=Abstracthttp://www.ncbi.nlm.nih.gov/pubmed/17878220?dopt=Abstracthttp://www.ncbi.nlm.nih.gov/pubmed/21698810?dopt=Abstracthttp://www.ncbi.nlm.nih.gov/pubmed/21698810?dopt=Abstracthttp://www.ncbi.nlm.nih.gov/pubmed/21698810?dopt=Abstracthttp://www.ncbi.nlm.nih.gov/pubmed/11739957?dopt=Abstracthttp://www.ncbi.nlm.nih.gov/pubmed/11739957?dopt=Abstracthttp://www.ncbi.nlm.nih.gov/pubmed/11238541?dopt=Abstracthttp://www.ncbi.nlm.nih.gov/pubmed/11238541?dopt=Abstracthttp://www.ncbi.nlm.nih.gov/pubmed/10650936?dopt=Abstracthttp://www.ncbi.nlm.nih.gov/pubmed/10650936?dopt=Abstracthttp://-/?-

7/29/2019 63987911

9/10

18. Schwarz EJ, Reginato MJ, Shao D, Krakow SL, Lazar MA: Retinoic acid

blocks adipogenesis by inhibiting C/EBPbeta-mediated transcription. Mol

Cell Biol 1997, 17:1552-61.

19. Mercader Josep, Ribot Joan, Murano Incoronata, Felipe Francisco,Cinti Saverio, Bonet Luisa M, Palou Andreu: Remodeling of White Adipose

Tissue after Retinoic Acid Administration in Mice. Endocrinology2006,

147:5325-32.20. Ziouzenkova Ouliana, Orasanu Gabriela, Sharlach Molly, Akiyama Taro E,

Berger Joel P, Viereck Jason, Hamilton James A, Tang Guangwen,

Dolnikowski Gregory G, Vogel Silke, Duester Gregg, Plutzky Jorge:

Retinaldehyde represses adipogenesis and diet-induced obesity. Nat Med

2007, 13:95-702.

21. Hauner H, Schmid P, Pfeiffer EF: Glucocorticoids and insulin promote the

differentiation of human adipocyte precursor cells in to fat cells. J Clin

Endocrinol Metab 1987, 64:832-835.

22. vara prasad Sakamuri SS, Prashanth Anamthathmakula, Kumar

Pavan Chodavarapu, Reddy Sirisha J, Giridhara Nappan V,

Vajreswari Ayyalasomayajula: A novel-genetically-obese rat model with

elevated 11-hydroxysteroid dehydrogenase type 1 activity in

subcutaneous adipose tissue. Lipids in Health Dis 2010, 9:132.

23. Paterson Janice M, Morton Nicholas M, Fievet Catherine,Kenyon Christopher J, Holmes Megan C, Staels Bart, Seckl Jonathan R,

Mullins John J: Metabolic syndrome without obesity: Hepatic

overexpression of 11-Hydroxystweroid dehydrogenase type 1 intransgenic mice. Proc Natl Acad Sci 2004, 101:7088-7093.

24. Tomlinson Jeremy W, Walker Elizabeth A, Bujalska Iwona J, Draper Nicole,

Lavery Gareth G, Cooper Mark S, Hewison Martin, Stewart Paul M: 11-

Hydroxysteroid Dehydrogenase Type 1: A Tissue-Specific Regulator of

Glucocortcoid Response. Endocrinology2004, 25:831-866.

doi:10.1186/1475-2891-10-70Cite this article as: Sakamuri et al.: Vitamin A decreases pre-receptoramplification of glucocorticoids in obesity: study on the effect ofvitamin A on 11beta-hydroxysteroid dehydrogenase type 1 activity inliver and visceral fat of WNIN/Ob obese rats. Nutrition Journal 2011 10:70.

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